The long term goals of this project are to understand the biochemistry, molecular biology and cell biology in humans of the two enzymes, bile acid CoA synthetase (hBAS) and bile acid CoA: amino acid N-acyltransferase (hBAT), which are responsible for the biosynthesis of the amino acid conjugates of bile acids. The naturally occurring C24 bile acids in human bile are found almost exclusively as their amino acid conjugates. These bile acid conjugates are by far the principal solutes of human bile and their secretion is the major determinant of bile flow. In bile and the small intestine, bile acid amino acid conjugates form mixed micellar complexes with water-insoluble lipid species such as phospholipids, monoacylglycerols, free fatty acids and cholesterol, thereby performing important physiological functions both in normal health and disease. To fully understand how hBAS and hBAT are involved in the synthesis of these conjugates in humans requires knowledge of the chemistry of these enzymes (their primary amino acid sequence), their genetic organization (nucleotide sequence of cDNAs encoding them and the gene(s) from which they were derived and their chromosomal localization), and the regions of each enzyme which are responsible for their catalytic properties. The specific aims of this application are (1) to characterize the chemical and biochemical properties of purified hBAT by determining whether it has Ping-Pong Bi Bi reaction mechanism, evaluating the importance of its three cysteine residues (cys-235, cys-372 and cys-373) to enzyme function, and examining the rules which govern its substrate specificity; (2) to purify hBAS, characterize its biochemical and chemical properties (as for hBAT) and raise specific polyclonal anti-hBAS antibodies in rabbits; (3) to use the hBAT cDNA to determine the heterogeneity of RNA sequences related to hBAT and the chromosomal localization of the hBAT gene. When these specific aims have been accomplished, it will be possible in future granting periods to clone hBAS and to further analyze which regions of the hBAS and hBAT are essential for enzyme catalytic function and substrate specificity.